Short-Term Plasticity in a Computational Model of the Tail-Withdrawal Circuit in Aplysia.

The tail-withdrawal circuit of Aplysia provides a useful model system for investigating synaptic dynamics. Sensory neurons within the circuit manifest several forms of synaptic plasticity. Here, we developed a model of the circuit and investigated the ways in which depression (DEP) and potentiation (POT) contributed to information processing. DEP limited the amount of motor neuron activity that could be elicited by the monosynaptic pathway alone. POT within the monosynaptic pathway did not compensate for DEP. There was, however, a synergistic interaction between POT and the polysynaptic pathway. This synergism extended the dynamic range of the network, and the interplay between DEP and POT made the circuit responded preferentially to long-duration, low-frequency inputs.

Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

Historical and idealized climate model experiments: an intercomparison of Earth system models of intermediate complexity

Both historical and idealized climate model experiments are performed with a variety of Earth system models of intermediate complexity (EMICs) as part of a community contribution to the Intergovernmental Panel on Climate Change Fifth Assessment Report. Historical simulations start at 850 CE and continue through to 2005. The standard simulations include changes in forcing from solar luminosity, Earth's orbital configuration, CO2, additional greenhouse gases, land use, and sulphate and volcanic aerosols. In spite of very different modelled pre-industrial global surface air temperatures, overall 20th century trends in surface air temperature and carbon uptake are reasonably well simulated when compared to observed trends. Land carbon fluxes show much more variation between models than ocean carbon fluxes, and recent land fluxes appear to be slightly underestimated. It is possible that recent modelled climate trends or climate–carbon feedbacks are overestimated resulting in too much land carbon loss or that carbon uptake due to CO2 and/or nitrogen fertilization is underestimated. Several one thousand year long, idealized, 2 × and 4 × CO2 experiments are used to quantify standard model characteristics, including transient and equilibrium climate sensitivities, and climate–carbon feedbacks. The values from EMICs generally fall within the range given by general circulation models. Seven additional historical simulations, each including a single specified forcing, are used to assess the contributions of different climate forcings to the overall climate and carbon cycle response. The response of surface air temperature is the linear sum of the individual forcings, while the carbon cycle response shows a non-linear interaction between land-use change and CO2 forcings for some models. Finally, the preindustrial portions of the last millennium simulations are used to assess historical model carbon-climate feedbacks. Given the specified forcing, there is a tendency for the EMICs to underestimate the drop in surface air temperature and CO2 between the Medieval Climate Anomaly and the Little Ice Age estimated from palaeoclimate reconstructions. This in turn could be a result of unforced variability within the climate system, uncertainty in the reconstructions of temperature and CO2, errors in the reconstructions of forcing used to drive the models, or the incomplete representation of certain processes within the models. Given the forcing datasets used in this study, the models calculate significant land-use emissions over the pre-industrial period. This implies that land-use emissions might need to be taken into account, when making estimates of climate–carbon feedbacks from palaeoclimate reconstructions.

From school to work: A comprehensive model of work-related motivation in the transition to working life.

Motivation is a core concept to understand work related outcomes and vocational pursuits. However, existing research mostly focused on specific aspects of motivation, such as goals or self-efficacy beliefs, while falling short of adequately addressing more complex and integrative notions of motivation. Advancing the current state of research, we draw from Motivational Systems Theory and a model of proactive motivation to propose a comprehensive model of work-related motivation. Specifically, we define motivation as a system of mutually related factors consisting of goals, emotions, and personal agency beliefs, comprised by capability beliefs and context evaluations. Adapting this model of motivation to the school-to-work transition, we postulate that this motivational system is affected by different social, personal, and environmental variables, for example social support, the presence of role-models, personality traits, and scholastic achievement. We further expect that students with more autonomous work-related goals, expectations of more positive emotional experiences in their future working life, fewer perceived barriers to their career development, and higher work-related self-efficacy beliefs would be more successful in their transition from school to work. We also propose that goal-directed engagement acts as a partial mediator in the relationship between motivation and a successful transition. Finally, we hypothesize that work-related motivation while in school will have meaningful effects on positive outcomes while in vocational training, as represented by more work engagement, higher career commitment, job satisfaction, and lower intentions to quit training. In sum, we advance the point that the adaptation of a broader concept of work-related motivation in the school-to-work transition would result in more powerful predictions of success in this transition and would enhance scientific research and interventions in career development and counselling practice.

Short-Term Plasticity in a Computational Model of the Tail-Withdrawal Circuit in Aplysia.

The tail-withdrawal circuit of Aplysia provides a useful model system for investigating synaptic dynamics. Sensory neurons within the circuit manifest several forms of synaptic plasticity. Here, we developed a model of the circuit and investigated the ways in which depression (DEP) and potentiation (POT) contributed to information processing. DEP limited the amount of motor neuron activity that could be elicited by the monosynaptic pathway alone. POT within the monosynaptic pathway did not compensate for DEP. There was, however, a synergistic interaction between POT and the polysynaptic pathway. This synergism extended the dynamic range of the network, and the interplay between DEP and POT made the circuit responded preferentially to long-duration, low-frequency inputs.

Eluding antibiotic resistance: capitalizing on antimicrobial peptides interaction with the lipid bilayer

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria. Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 to 15 examples of modified natural and synthetic AMPs in clinical development. But most of these agents are against Gram-positive bacteria. Therefore, there is unmet medical need for antimicrobials used to treat infections caused by Gram-negative bacteria. In this study, we show that a pro-apoptotic peptide predominantly used in cancer therapy, (KLAKLAK)2, is an effective antimicrobial against Gram-negative laboratory strains and clinical isolates. Despite the therapeutic promise, AMPs development is hindered by their susceptibility to proteolysis. Here, we demonstrate that an all-D enantiomer of (KLAKLAK)2, resistant to proteolysis, retains its activity against Gram-negative pathogens. In addition, we have elucidated the specific site and mechanism of action of D(KLAKLAK)2 through a repertoire of whole-cell and membrane-model assays. Although it is considered that development of resistance does not represent an obstacle for AMPs clinical development, strains with decreased susceptibility to these compounds have been reported. Staphylococci resistance to DAP was observed soon after its approval for use and has been linked to alterations of the cell wall (CW) and cellular membrane (CM) properties. Immediately following staphylococcal resistance, Enterococci resistance to DAP was seen, yet the mechanism of resistance in enterococci remains unknown. Our findings demonstrate that, similar to S. aureus, development of DAP-resistance in a vancomycin-resistant E. faecalis isolate is associated with alterations of the CW and properties of the CM. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus.

A convolution model for energy transport in a therapeutic fast neutron beam

A three-dimensional model has been proposed that uses Monte Carlo and fast Fourier transform convolution techniques to calculate the dose distribution from a fast neutron beam. This method transports scattered neutrons and photons in the forward, lateral, and backward directions and protons, electrons, and positrons in the forward and lateral directions by convolving energy spread kernels with initial interaction available energy distributions. The primary neutron and photon spectrums have been derived from narrow beam attenuation measurements. The positions and strengths of the effective primary neutron, scattered neutron, and photon sources have been derived from dual ion chamber measurements. The size of the effective primary neutron source has been measured using a copper activation technique. Heterogeneous tissue calculations require a weighted sum of two convolutions for each component since the kernels must be invariant for FFT convolution. Comparisons between calculations and measurements were performed for several water and heterogeneous phantom geometries. ^

Immunochemical interactions of peptides with site-directed polyclonal immunoglobulin G

The objective of this study was to investigate the immunochemical nature of the polyclonal immune response to the 14mer peptide TINKEDDESPGLYG and to identify interactions among antibodies to more than one epitope. Two groups of rabbits were immunized with the 14mer peptide and a Keyhole Limpet hemocyanin (KLH) carrier, but with KLH attached either to the 14mer's N- or C-terminus. Two approximate epitopes were mapped by an antibody-capture enzyme-linked immunosorbent assay method using antiserum obtained when KLH was oriented on the C-terminus of the 14mer. A precise mapping of the epitopes performed with inhibition enzyme immunoassays (iEIAs) resulted in an N-terminal 6mer epitope TINKED and a C-terminal 10mer epitope EDDESPGLYG. The epitopes overlapped by two amino acids. IEIAs and iEIAs incorporating antibody-blocking peptides indicated that the two anti-epitope antibody fractions did not interfere with one anothers' epitope binding. It was postulated that the anti-TINKED and anti-EDDESPGLYG antibody fractions individually bind their respective hydrophobic epitope "core" region at the N- or C-terminal of peptide TINKEDDESPGLYG, while sharing the two hydrophilic overlap amino acids. This antibody "lap joint" binding interaction can be accomplished by each of the anti-epitope antibodies binding an opposite side of the epitope overlap region in the shallow periphery of its binding site. ^

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

A cardiovascular mathematical model of graded head-up tilt

A lumped parameter model of the cardiovascular system has been developed and optimized using experimental data obtained from 13 healthy subjects during graded head-up tilt (HUT) from the supine position to [Formula: see text]. The model includes descriptions of the left and right heart, direct ventricular interaction through the septum and pericardium, the systemic and pulmonary circulations, nonlinear pressure volume relationship of the lower body compartment, arterial and cardiopulmonary baroreceptors, as well as autoregulatory mechanisms. A number of important features, including the separate effects of arterial and cardiopulmonary baroreflexes, and autoregulation in the lower body, as well as diastolic ventricular interaction through the pericardium have been included and tested for their significance. Furthermore, the individual effect of parameter associated with heart failure, including LV and RV contractility, baseline systemic vascular resistance, pulmonary vascular resistance, total blood volume, LV diastolic stiffness and reflex gain on HUT response have also been investigated. Our fitted model compares favorably with our experimental measurements and published literature at a range of tilt angles, in terms of both global and regional hemodynamic variables. Compared to the normal condition, a simulated congestive heart failure condition produced a blunted response to HUT with regards to the percentage changes in cardiac output, stroke volume, end diastolic volume and effector response (i.e., heart contractility, venous unstressed volume, systemic vascular resistance and heart rate) with progressive tilting.

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs

Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

True four-dimensional analysis of thoracic aortic displacement and distension using model-based segmentation of computed tomography angiography.

Previous analyses of aortic displacement and distension using computed tomography angiography (CTA) were performed on double-oblique multi-planar reformations and did not consider through-plane motion. The aim of this study was to overcome this limitation by using a novel computational approach for the assessment of thoracic aortic displacement and distension in their true four-dimensional extent. Vessel segmentation with landmark tracking was executed on CTA of 24 patients without evidence of aortic disease. Distension magnitudes and maximum displacement vectors (MDV) including their direction were analyzed at 5 aortic locations: left coronary artery (COR), mid-ascending aorta (ASC), brachiocephalic trunk (BCT), left subclavian artery (LSA), descending aorta (DES). Distension was highest for COR (2.3 ± 1.2 mm) and BCT (1.7 ± 1.1 mm) compared with ASC, LSA, and DES (p < 0.005). MDV decreased from COR to LSA (p < 0.005) and was highest for COR (6.2 ± 2.0 mm) and ASC (3.8 ± 1.9 mm). Displacement was directed towards left and anterior at COR and ASC. Craniocaudal displacement at COR and ASC was 1.3 ± 0.8 and 0.3 ± 0.3 mm. At BCT, LSA, and DES no predominant displacement direction was observable. Vessel displacement and wall distension are highest in the ascending aorta, and ascending aortic displacement is primarily directed towards left and anterior. Craniocaudal displacement remains low even close to the left cardiac ventricle.